WO2006098769A2 - Multipathogen and monopathogen protection against the bacterial and viral infections associated with biological warfare, cardiac diseases, and cancers - Google Patents

Abstract

Methods for developing vaccines and treatments against pathogens associated with biological warfare, cardiac disease, cancer, and emerging infectious diseases is provided. Amino acid sequences of immunogenic peptides and compositions comprising them are also described. Also described are antibodies raised against the immunogenic peptides of the present invention.

Description

MULTIPATHOGEN AND MONOPATHOGEN PROTECTION

AGAINST THE BACTERIAL AND VIRAL INFECTIONS

ASSOCIATED WITH BIOLOGICAL WARFARE, CARDIAC

DISEASES, AND CANCERS

This invention was made with partial Government support under contract number W911NF-04-C-0046 awarded by DARPA. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

This invention relates to vaccines and treatments, as well as methods of making vaccines and treatments, to protect against the detrimental effects of biological warfare, heart diseases, cancers, and emerging infectious disease pathogens. Viruses and bacteria are responsible for a vast number of diseases affecting humans and other animals. In addition to the pathogens encountered in everyday life, there are some pathogens that can be weaponized and used in biological warfare. These include, but are not limited to, Variola virus ('smallpox virus), Bacillus anthracis, Yersinia pestis, Brucella suis, Francisella tularensis, Burkholderia mallei, encephalitis, and hemorrhagic fever viruses.

Because pathogens used in biological warfare are typically encountered in the field before they can be identified, it is important that preventative measures protect against a broad spectrum of potential pathogens. The vaccines currently available that protect against individual pathogens are, therefore, not as useful as a vaccine that would be protective against a broad spectrum of pathogens.

In addition to the pathogens that can be used in biological warfare, more commonly encountered pathogens have been found to cause some of the most prevalent diseases that debilitate humans every day. For example, cardiac disease has been tied to the involvement of microbes, such as, but not limited to, cytomegalovirus and other herpesviruses, coxsackieviruses, echoviruses, hepatitis viruses, influenza viruses, chlamydia, Streptococcus pneumoniae, and Staphylococci. Similarly, certain cancers have also been shown to be associated with specific viruses and bacteria, including, but not limited to, human papilloma viruses, Epstein Barr virus, hepatitis B virus, human lymphotrophic viruses, retroviruses, reoviruses, human herpesvirus 8 (HHV8), influenza viruses, and coxsackieviruses, Helicobacter pylori, Citrobacter, Salmonella, etc.

Like the pathogens used in biological warfare, the pathogens related to cardiac disease and cancer cover a broad spectrum. Vaccines against individual pathogens, then, are not as effective in preventing cardiac disease or cancer as vaccines that target a correspondingly broad spectrum of pathogens.

The existing therapies are often not effective in preventing and treating diseases caused by pathogens. There is, therefore, a compelling need to develop new more effective compositions and methods for treatment and prevention of diseases caused by the pathogens of biological warfare, as well as for more common diseases, such as cardiac diseases, cancers and emerging infectious diseases.

BRIEF SUMMARY OF THE INVENTION

The invention fulfills this need in the art by providing methods of developing vaccines, antibodies and other inhibitors of pathogens.

Accordingly, in one aspect, the present invention provides a method of developing vaccines, antibodies and other inhibitors of pathogens to be used in subjects in need thereof. The method comprises: (a) identifying at least one pathogen of interest; (b) obtaining a sequence of at least one protein produced by the pathogen; (c) identifying at least one target region of the sequence comprising an allosteric binding site or an active site of the protein; (d) selecting at least one portion of the target region that is accessible by a solvent to obtain at least one candidate sequence; (e) synthesizing candidate peptides having the candidate sequences; and (f) screening the candidate peptides for an immunogenic activity to identify at least one immunogenic peptide.

In one embodiment, the protein in the step (b) is selected from a group consisting of virulence enhancing enzymes, including, but not limited to, proteases and protease homologues and virulence factors. The identified immunogenic peptides may be administered to a patient directly. Alternatively, the immunogenic peptides may be injected into an animal to raise antibodies, which later may be used to treat a patient. The antibodies may be monoclonal or polyclonal antibodies. In one embodiment, step (d) further comprises comparing the candidate sequences with animal protein sequences, if the subject is an animal, or with the human protein sequences, if the subject is a human, and excluding the candidate sequences having a predetermined level of homology with animal or human proteins from further method steps. In one embodiment, the predetermined level of homology is at least 25%. In another embodiment, step (e) further comprises conjugating each of the candidate peptides to a carrier protein to obtain conjugated peptides and step (f) comprises screening the conjugated peptides for an immunogenic activity.

In another aspect, the present invention provides compositions comprising at least one immunogenic peptide identified in accordance with the methods of the present invention or at least one antibody raised against such peptide. The compositions of the present invention may optionally have additional ingredients. In one embodiment, additional ingredients are protease inhibitors or inhibitors of apoptosis. In another embodiment, the composition comprises an antibody obtained in accordance with the methods of the present invention and the additional active ingredient is an antimicrobial agent.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure IA depicts SDS-PAGE of B. anthracis culture supernatant (BACS) fractions separated on size exclusion column. Figures IB, 1C, and

IF represent Western blots, with Figure IB using specific antisera a-M4EL, Figure 1C, left panel, using BACS with a-M4AC, Figure 1C, right panel using BACS with a-M4EP, and Figure IF using BACS with a-M9Coll. Zymograms are depicted in Figures 1C and IE, with the caseinolytic and gelatinolytic activities of BACS, respectfully. The molecular mass (KDa) of the marker proteins are indicated by arrows. In Figure IA, symbol "s" denotes BACS, and numbers above the blot correspond to column fractions. In Figure IE, different amounts of BACS were loaded on a gel (15 μl, 7μl and 3 μl, from left to right). Figure 2 depicts post-exposure efficacy of hyperimmune rabbit sera in mice challenged with B. anthracis (Sterne). Treatment with sera and ciprofloxacin was initiated 24 hours post exposure and continued for 10 days, once daily. In Figure 2A, 5 mg/kg of serum was administered with ciprofloxacin; in Figure 2B, only serum was administered; in Figure 2C, 25 mg/kg of serum was administered with ciprofloxacin.

Figures 3A - 3H provide structural representations of the target region comprising an allosteric binding site or an active site of several chosen metalloproteases. The light grey shading represent the alignment of amino acid residues of the structure to the protease amino acid residues from the target region. The structure code and sequence is listed on the top and the corresponding target sequence taken from the protease is listed on the bottom. The black residues are surface residues of the candidate metalloproteases, according to the RasMol program, Open Source graphics program intended for the visualization of proteins, nucleic acids and small molecules.

Figure 4 demonstrates survival of vaccinated ("Antrax:A.2") and non-vaccinated ("KLH") mice challenged with 5 xlO⁷ spores of Sterne Strain B. anthracis. The vaccinated mice were immunized with a combination of peptides as described in Tables 2-3 conjugated to Keyhole

Limpet Hemocyanin (KLH); and non-vaccinated control mice were treated with KLH only.

Figure 5 represents protective effect of the individual peptides (Fig.5, A-F), as well as protective effect of the following combinations: Combination T.I (Fig.5, G), which contains the peptide sequences

DELNSIVENN, TSMVHEPNFD, EGHLLSPLLD, NRLMTPASTN, ETIVKDTDRD, YWFGKDALEL, Combination T.2 (Fig.5, H), which contains the peptide sequences NRLMTPASTN, ETIVKDTDRD, YWFGKDALEL, IHLSVQANA V, THFGEHPSLKI, DAMSTNKFGV, and Combination T.3 (Fig.5, I), which contains the peptide sequences

DELNSIVENN, TSMVHEPNFD, EGHLLSPLLD, IHLSVQANAV, THFGEHPSLKI, DAMSTNKFGV Balb/c mice were immunized with 10 μg of peptide for individual vaccinations and 60 μg of total peptide for combinations (six peptides at 10 μg per peptide). Mice were challenged with IOLD5O of Francisella tularensis LVS i.p.

Figure 6 represents the protective effect of antibodies raised from nearly identical sequences in anthrax and plague, which resulted in 100% survival in late-stage treatment of anthrax (Fig.6, A) and plague (Fig.6, B).

The concentration of antibodies used was 10 mg/kg to treat anthrax, 20 mg/kg to treat plague, and ciprofloxacin was used at 50 mg/kg. Mice were challenged with 5x10⁷ spores/mouse of Bacillus anthracis Sterne strain or 6xlO⁷ organisms/mouse of Yersinia pestis KIM5-3001.1. The resulting p- value for the groups providing 100% survival was p<0.01 compared to untreated mice.

Figure 7 demonstrates the protective effect against anthrax using antibodies raised against a number of peptide sequences representing epitopes in proteases from B. anthracis. Sequences comprising the active center of metal loproteases M4, M6, M32, M34, and M60 were identified (see table 1) and antibodies against these sequences were raised in rabbits. The antibodies were tested against anthrax individually and in combination with ciprofloxacin. Antibodies were used in a concentration of 10 mg/kg and the concentration of ciprofloxacin was 50 mg/kg. Mice were infected with B. anthracis Sterne strain with a challenge dose of 5 x 107 spores/mouse.

Treatment was initiated 24 hours after infection and continued for 10 days. Figure 7A demonstrates 60% survival using anti-M32 antibodies in combination with ciprofloxacin versus 10% survival of ciprofloxacin only treated mice. Figure 7B demonstrates 60% survival using anti-M60 antibodies in combination with ciprofloxacin versus 10% survival of ciprofloxacin only treated mice. Figure 7C demonstrates 100% survival using a combination of anti-M4, anti-M6, anti-M32, and anti-M34 antibodies in combination with ciprofloxacin versus 0% survival of ciprofloxacin only treated mice.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention encompasses methods of developing treatments for the pathogens of biological warfare, cardiac diseases, cancers, and emerging infectious diseases. Accordingly, in one aspect the invention provides a method of developing vaccines, antibodies and other inhibitors of pathogens to be used in animal and/or human patients. The method comprises the steps of: (a) identifying at least one pathogen of interest; (b) obtaining a sequence of at least one protein produced by the pathogen; (c) identifying at least one target region of the sequence comprising an allosteric binding site or an active site of the protein; (d) selecting at least one portion of the target region that is accessible by a solvent to obtain at least one candidate sequence; (e) synthesizing candidate peptides having the candidate sequences; and (f) screening the candidate peptides for an immunogenic activity to identify at least one immunogenic peptide.

As used herein, the term "peptide" refers to a chain of amino acids, wherein the α-carboxyl group of one amino acid is joined to the cc-amino group of another amino acid by a peptide bond. In peptides of the present invention, the chain of amino acids is longer than one amino acid, but shorter than 100 amino acids.

The term "protein" refers to one or more chains of amino acids that either have a catalytic function (referred to as an enzyme) or a structural function (used to build the microbe). This invention does not limit length of amino acid chains in proteins and their subunits. The term "allosteric binding site" refers to a portion of a protein sequence that is located outside of the protein's active site and is not involved in a substrate binding (i.e. the sequence is simply structural in the protein), but the sequence is predicted as an epitope that an antibody would bind to. For example, allosteric binding sites may include sequences that are solvent exposed, and predicted to bind to major histocompatability complexes class one or class two (MHC I or MHC II, respectively). The predictions may be made using epitope prediction software, which uses patterns of peptides that are known to bind histocompatability complexes.

The term "solvent" refers to any liquid, including, but not limited to, water, aqueous solutions, organic liquids, and organic solutions.

The term "immunogenic activity" refers to a specific immune response (cellular or humoral) in the host induced by a peptide or another antagonist. Those skilled in the art would appreciate that a broad range of in vitro and in vivo techniques are routinely used to determine immunogenic activity. For example, the immunogenic activity may be measured using enzyme-linked immunosorbent assay (ELISA) with a minimal cut off of two standard deviations from the mean of the control optical density value. A lymphocyte proliferation assay may also be used to detect immunogenic activity. In lymphocyte proliferation assays, detection is measured in counts per minute in a ratio where the stimulation index (SI) = (cpm experimental/ cpm background unstimulated) and a significant value is in the range from 3 to 5. Any other in vitro methods that confirm binding of an antibody to the peptide sequence or antibody to the native protein, from which the peptide was derived from, may also be used. In vivo screening may be carried out, for example, by measuring survival rate in animals.

The term "antibody binding" is defined as the ability of an antibody to specifically bind a peptide sequence. Specific binding is defined as greater than 10⁵ M^'1.

The term an "immune response" (or an "immunogenic response") refers to a reaction in a host to an administered substance, wherein specific antibodies are produced or other cells of the immune system are specifically stimulated. A "protective immune response" is an immune response in which the antibodies and cellular immune reactions produced are protective against a disease or diseases. In one embodiment, the immunogenic response is defined as an increased survival by at least 30% as compared to a non- vaccinated group. A "vaccine" is a substance administered to a human or other animal that produces an immune response and protects the human or other animal from a disease. Vaccines may be administered before exposure of human or other animal to anthrax.

The terms "passive immunization" or "passive immunity" refer to a transfer of antiserum from one animal to another or from an animal to a human to achieve immunity against a disease in the recipient. Passive immunization may be performed before or after the recipient has been exposed to the disease. The term "therapy" refers to any course of action, including the administration of substances, to a patient suffering from a disease to alleviate the symptoms of the disease. For the purposes of the present invention, the term "antimicrobial" is used generally to include any agent that is harmful to microbes, including agent with antibacterial, antifungal, antialgal, antiviral, antiprotozoan, and other such activity. The term "antibiotic" is used in the present invention to refer to an antibacterial agent.

For the purposes of the present invention, the term "conservative modification" refers to a change in the amino acid composition of a peptide that does not substantially alter its activity. Such conservative modifications are known to those skilled in the art and may include substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids, e.g., often, less than 5%, in the amino acid sequence.

For example, conservative modification may comprise of substitution of amino acids with other amino acids having similar properties such that the substitutions of even critical amino acids does not substantially alter immunogenic activity of the peptide. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),

Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W) (see also, Creighton, 1984, Proteins, W. H. Freeman and Company). A conserved modification may also include mutating the amino acid residues that are not surface exposed in the native protein. These residues should not interact with the antibody and so changing them should still produce an antibody of equivalent affinity. Another example of conservative modification is adding amino acids to the N or C terminus that are not existent in the native protein sequence, but would increase antibody production. Peptides obtained by such additions are often referred to as constructs.

Generally, the ability of bacteria to cause destruction of tissues, degradation of immunoglobulins and cytokines, the release of inflammatory mediators or the activation of host proteolytic enzymes, is attributed to a wide variety of secreted proteolytic enzymes (also referred to as proteases) (Supuran et al., 2002). Proteases are enzymes that hydrolyze peptide bonds in biomolecules (Supuran et al, 2002). Accordingly, in one embodiment, amino acid sequences of proteases are analyzed to identify target regions comprising their allosteric binding sites and active sites (see Example 1). Although not wanting to be bound by a theory, inventors believe that binding of an antibody to or around the active center or another binding site of a protein will prevent the binding of the substrate and annihilate the function of the protein.

Metalloproteases (MPs) are proteases, in which the nucleophilic attack on the peptide bond is mediated by water molecule coordinated to a divalent metal ion or bridged to a dimetalic center (Supuran et al, 2002). Some of MPs from several bacterial species are known to be capable of causing massive internal hemorrhages and other death-threatening pathologies (Supuran et al., 2002; Sakata et al., 1996; Shin et al., 1996; Miyoshi et al., 1998; Okamoto et al., 1997). Accordingly, in one embodiment, sequences of several MPs are analyzed to identify target regions comprising their allosteric binding sites and active sites (see, for instance, Examples 1 and 2).

For example, the sequence of thermolysin, an extracellular metal loendoprotease secreted by the Bacillus anthracis, was analyzed. Thermolysin and related M4 family proteases have an active site, which contains a Zn(II) ion coordinated by two histidine residues belonging to a His-Glu-Xaa-Xaa-His (HEXXH) active site motif (Supuran, 2002). Another

MP family, M9, includes bacterial collagenases, collagen degrading enzymes (Supuran, 2002). Proteases that belong to the M9 and many other protease families also possess the HEXXH active site motif (Supuran, 2002).

Other groups of proteases, in general, and MPs, in particular, may have different conserved amino acid motifs associated with the active site.

For example, three motifs, [S,G,A]-H-X-D-X-V, G-X-X-D, and X-E-E, have been suggested to be involved in zinc coordination at the active site of certain metal loexoproteases (Biagini, 2001). Each of the identified target regions were analyzed to determine which portions of the target regions (candidate sequences) are accessible to a solvent and/or are predicted to be located on an outer surface of a tertiary structure of the protein. In one embodiment, the candidate sequences comprise at least 6 amino acids. In another embodiment the candidate sequences comprise less than 100 amino acids.

Those skilled in the art would appreciate that a number of computer programs may be used to predict surface regions, regions accessible to a solvent, antigenicity, or hydrophilicity of a stretch of amino acid sequence. For example, the MEROPS database [Barrett AJ, 2004] provides wealth of information on proteases and their inhibitors. The data is categorized into individual summary pages of proteases, including sequence information and organism source. Individual proteases are then grouped into families and individual families grouped into clans (each with its own summary record), based on statistical sequence similarity (Rawlings et al., 2004). For each protease family, MEROPS provides multiple alignments of the protease sequences within the family and a distribution chart of the protease family among the major taxonomic groups, including bacteria, viruses, animals, and plants (Rawlings et al., 2004). In one embodiment, when target region comprises an allosteric binding site or an active site and it is determined that the candidate sequence is not located on the surface of the protein and/or is not accessible to a solvent, the following steps are implemented.

First, the candidate sequence is shifted up to about 20 amino acids in either direction along the linear amino acid sequence until its amino acid residues are located on the surface of the protein and/or are solvent accessible (referred to as an "exposed candidate sequence"). Second, if such shift does not result in an exposed candidate sequence and other active or allosteric site(s) exist(s) in another part of the linear sequence, then the sequence is analyzed for exposed candidate sequences around those sites.

Third, if the first and the second steps do not lead to an identification of at least one exposed candidate sequence, alternative candidate sequence(s) is(are) identified through visualization of the area around the active center and around allosteric site to identify surface fragments. The alternative sequences may or may not contain amino acids from the active center motif. Candidate peptides comprising candidate sequences are then synthesized and tested for an immunogenic activity in vitro or in vivo.

In one embodiment, step (d) of the instant method further comprises comparing the candidate sequences with animal protein sequences, if the subject is an animal, or with human protein sequences, if the subject is a human, and excluding the candidate sequences having a predetermined level of homology with the animal or human proteins from further method steps to avoid cross-reactivity with similar proteins of the subject. A number of methods exist that allow a homology analysis. One such method is the Basic Local Alignment Search Tool (BLAST), which performs sequence similarity searches against a variety of sequence databases, returning a set of graphed alignments between the query and database sequences (Wheeler, 2004). In one embodiment, the selected protease sequences were BLASTed against the human Reference Sequence

(RefSeq) collection of proteins (Pruitt et al., 2003) to confirm that they do not express significant homology to any human protein (Altschul et al., 1997). In one embodiment, the predetermined level of homology is at least about 25%. In one embodiment, step (e) further comprises covalently conjugating each of the candidate peptides to a carrier protein, such as Keyhole Limpet Hemocyanin (KLH), to obtain conjugated peptides and step (f) comprises screening the conjugated peptides for an immunogenic activity. KLH a vehicle for delivery the immunogenic peptides of the present invention and prevents their degradation in the blood circulation.

In another aspect, the present invention provides a composition comprising (i) at least one immunogenic peptide identified according to the method described above or (ii) at least one antibody raised against such immunogenic peptide. The invention also provides a method of preventing or treating infections caused by a pathogen in a subject. The method comprises administering to the subject one of the compositions of the present invention. The subject may be a human or an animal.

Antibodies of this invention include both polyclonal and monoclonal antibodies. Those skilled in the art would know how to raise antibodies against identified immunogenic peptides. For example, in vitro 3-D splenocyte-based tissue systems may be used to raise antibodies. Alternatively, laboratory animals may be used for in vivo generation of antibodies. Furthermore, some companies, such as Invitrogen (CA), take orders on production of monoclonal and polyclonal antibodies utilizing standardized methods against peptides identified by a customer.

Polyclonal antibodies may be prepared, for example, according to the method described in Leenaar and Hendricksen, 2005. In one embodiment, one or a plurality of the immunogenic peptides identified according to the methods of the present invention or their conjugates are injected into laboratory animals capable of raising large amounts of antibody, such as, but not limited to rabbits, sheep, cows, horses, mice, goats, monkeys, rats, etc. intraperitoneal Iy, subcutaneously, intramuscularly, or into the ear vein, groin, etc. The immunogenic peptides may be administered together with

Freund's complete or incomplete adjuvants. The immunogenic peptide is usually administered every several weeks. The titer can be elevated by the booster injection. Blood is periodically collected to confirm the titer elevation by ELISA, etc. After the final immunization, the blood is collected from immunized animals to obtain antisera. Antisera are purified by salting out, ion exchange chromatography, HPLC, etc. to obtain an IgG fraction. The antibody can be further purified by affinity chromatography using the immobilized peptides. The purified polyclonal antibodies are administered to a subject to provide passive immunization or as a therapy for a disease. A monoclonal antibody may be prepared, for example, according to the method described Roque et al. (2004). In one embodiment, animals are immunized with the immunogenic peptide of this invention, and, after the final immunization, spleen or lymph node is excised from immunized animals. Antibody-producing cells contained in the spleen or lymph node are fused with myeloma cells using a fusing agent such as polyethylene glycol, to prepare hybridomas. Desired hybridomas are screened and cultured to prepare a monoclonal antibody from the culture supernatant. The monoclonal antibody is purified by salting out, ion exchange chromatography, FIPLC, etc. to obtain an IgG fraction. The resulting fraction can be further purified by affinity chromatography using the immobilized peptide. Monoclonal antibodies of the present invention may be chimeric antibodies comprising human constant regions.

In alternative embodiments of the invention, single pathogens are targeted or multiple pathogens are targeted with the peptide or peptides developed in accordance with the methods of the present invention.

The invention further encompasses treatments for the pathogens and diseases of biological warfare, cardiac disease, cancer, and emerging infectious diseases. In these treatments, the peptide or peptides determined by the methods of the invention are administered to a patient and the effects of the disease are alleviated.

The diseases of biological warfare include, but are not limited to, anthrax, smallpox, human monkeypox, plague, tularemia, glanders, melioidosis, brucellosis, botulism, tetanus, Ebola virus infection, Marburg virus, Lassa fever, Bolivian hemorrhagic fever, Argentinean hemorrhagic fever, Venezuelan equine encephalomyelitis, etc.

Cardiac diseases include, but are not limited to, atherosclerosis, stenosis of the blood vessels, restenosis of the blood vessels, myocardial infarction, myocarditis, pericarditis, acute and chronic cardiomyopathies, hypertension, etc.

Cancers include, but are not limited to, carcinomas, leukemias, sarcomas, Burkitt's lymphoma, nasopharyngeal carcinoma, papilloma, Kaposi's sarcoma, hepatocellular carcinoma, Hodgkin's Disease, myeloid leukemia, bladder cancer, brain cancer, head and neck cancer, kidney cancer, lung cancers, such as small cell lung cancer and non-small cell lung cancer, myeloma, neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, colon cancer, cervical carcinoma, breast cancer, epithelial cancer, and gastric cancer.

The invention also encompasses treatments for the pathogens of emerging infectious diseases. These include, but are not limited to,

Salmonella, Shigella, Escherichia coli, Vibrio cholerae, Haemophilus influenzae, Streptococcus pneumoniae, and Neisseria meningitides.

The present invention further relates to compositions, methods, processes, and systems for identifying and validating target proteases and/or their specific amino acid sequences useful as therapeutic and prophylactic targets for modulating diseases associated with microbes as well as to the principles and methods of accelerated drug development. The term "microbial" includes any microorganism or microorganism-like agent, including, but not limited to, bacteria, fungi, mycoplasma, rickettsia, and viruses.

The invention further provides a single peptide based or multi- peptide based vaccines against a broad range of diseases caused by pathogens for prophylaxis, or specific immunoglobulins for both prophylaxis and therapy. In one embodiment, there are multiple targets on a protein corresponding to different binding sites which are targeted by different sets of peptides to completely annihilate function of the protein. Accordingly, in this embodiment a plurality of peptides is provided to a subject simultaneously or is used to generate antibodies. The present invention demonstrates therapeutic efficacy of the peptide vaccines obtained in accordance with the methods of the present invention for protection against infections caused by B. anthracis (Examples 5, 6), Y.pestis (Example 6), and F. tularensis (Example 7). The present invention also demonstrates effective treatment of infections caused by B. anthracis (anthrax) (Examples 8, 9) and Y.pestis (plague) (Example 8) using antibodies raised according to the methods of the present invention. The antibodies may be used either alone or in a combination with antimicrobial agents.

These results demonstrate that the methods of the present invention provide immunogenic peptides and antibodies effective in prevention and treatment of infections caused by three different pathogens. The inventors, therefore, believe that the method of the present invention may similarly be used to develop vaccines against and treatments for diseases caused by any other pathogens. In one embodiment, the administering of the immunogenic peptide(s) of the present invention create(s) an immunogenic response in the subject within about 1 year. In another embodiment, the immunogenic response occurs between three and nine weeks after the vaccination with the peptide(s). In still another embodiment, the administering of the antibody(ies) ot the present invention protects the subject against natural and genetically engineered pathogens by administering the antibody(ies) either pre- or post-exposure, where pre-exposure is a time no greater than 12 hours before exposure. In one embodiment, the immunogenic peptide(s) or the antibody(ies) of the present invention is(are) administered in a form of a pharmaceutical composition. Those skilled in the art would recognize that a number of ingredients are routinely included in pharmaceutical compositions to adapt them to various forms of administration, to improve their stability, obtain higher efficiency, and so on. Accordingly, a pharmaceutical composition of the present invention may further include one or more pharmaceutical ingredients, including, but not limited to, active ingredients, including antimicrobial agents, and inactive ingredients, such as water and other inorganic and organic solvents, adjuvants, preservatives, pharmaceutical carriers, excipients, diluents, and stabilizers, to name a few.

Adjuvants include, but are not limited to alum, killed Bordetella pertussis, oil emulsion, Freund's complete or incomplete adjuvant, or any other material that can be added to an antigen to increase its immunogenicity.

The active ingredient added to or co-administered with pharmaceutical compositions of the present inventions may be an antimicrobial agent. In one embodiment, the pharmaceutical composition comprises the antibody(ies) of the present invention and an antimicrobial agent, such an antibiotic ciprofloxacin.

In one embodiment, the pharmaceutical composition comprises the immunogenic peptide(s) of the present invention and the composition is administered before the subject is exposed to a pathogen. In another embodiment, the pharmaceutical composition comprises the antibody(ies) of the present invention and the composition is administered before or after the subject is exposed to a pathogen. Specific dosing ranges and methods of administration are different for various types of vaccines, therapeutic preparations, and diseases. Optimal concentrations and regimens are determined empirically for each vaccine and therapeutic preparation based on prophylactic and therapeutic efficacy by dosing regimes known in the art. For example, methods of administering peptide(s) for vaccination may include oral, intranasal, subcutaneous, intravenous, intramuscular, intraperitoneal, or jet injector administration and doses from about 10 μg to about 1 g may be used.

Examples of methods of administering antibodies for treatment of anthrax infection may include intravenous or subcutaneous administration.

In one embodiment, the amount of antibody administered to the patient is from 100 mg to 1 g. The amount of antibody may also be determined on a per weight basis. Doses of antibodies may range from 0.1 mg/kg to 100 mg/kg or more. In one embodiment, the antibody is administered at 10 mg/kg.

In one embodiment of the invention, antibodies are administered intravenously or subcutaneously, and antibiotics are administered orally, intravenously, or subcutaneously. Injectable forms of the antibiotics or antibodies may be administered intravenously or subcutaneously, while oral administration may involve administration of tablets, solutions, lozenges, etc.

In another aspect of the present invention, compositions comprising specific peptides are disclosed. In one embodiment, a composition useful for protection against infections caused by B. anthracis is provided. The composition comprises at least one peptide comprising the sequence selected from the group consisting of GTLHEIAHGYQA, DVIGHELTHAVT,

GAVGVFAHEYGH, ELFRHEFTHYLQ, VIGHELTHAVTE, EYDTQYSGHGE, ELFRHEFTHYLQ, SAIPGTSEHQT, LILFEPANFNSM, DVIGHELTHAVTE, FGTIHECGHAVY, EGFIHEFGHAVD, EEVGLRGAKTSAN, ADYTRGQGIETY, MGIVHETGHARY, VAGHLDEVGFMI, WGCLHEIAHGYQ,

EEVGLRGAKTS, LVSIAGPISN, WGTLHEIAHGYQAG, WGTLHEIAHGYQA, HRMKDGSMFGS, VHGLNDWNVK, WLHQGGHGG, EIDNGKKYYLL, YNDKLPLYIS, DGAVGVFAHEYGH, EGFIHEFGHAVDD, VPDRDNDGIPDS, FLSPWISNIH, NEPNGNLQYQGK, DQQTSQNIKNQ, DKLPLYISNPNY,

NSRKKRSTSAGPT, VYYEIGK, HQSIGSTLYNK, DHRTVLELYAP, AND conservative modifications thereof.

In another embodiment, a composition useful for protection against infections caused by Yersinia pestis is provided. The composition comprises at least one peptide comprising the sequence selected from the group consisting of FNLAD VAICIG AAL, NAGYYVTPNAKV, GRQTFTHEI, GHALGLSHP, SRHHWGSDLDI, LMGIVHETGHARYE, MGIVHETGHARY, EMDIQRSSSECGIFS, SCEGSQFKLFD, SMGGMAASGGYWIS, AKGYWVENGEI, TTIQVDGSEKK,

SQDGNNHQFTT, DPQWKYSQETA, DNFMKDVLRLIEQ, EQYVSSHTHAMK, SPYGPEARAELSSR, GNMDDYDWMNEN, WVRAHDNDEHYM, FNLADVAICIGAAL, SRHHWGSDLDIYDP,

WGCLHEIAHGYQGGF, LILFEPANFNSMG, and conservative modifications thereof.

In still another embodiment, a composition useful for protection against infections caused by Francisella tularensis is provided. The composition comprises at least one peptide comprising the sequence selected from the group consisting of GYGDFKLLAA, KPQFTSYSVVD, DELNSIVENN, TSMVHEPNFD, EGHLLSPLLD, TAYHEAGHAI,

STGGSKMYVK, NRLMTPASTN, ETIVKDTDRD, YWFGKDALEL, EKLQTTHFSI, IHLSVQANAV, GKFSLDVDFT, THFGEHPSLKI,

DAMSTNKFGV, VGSQNSSNSNR, and conservative modifications thereof. In further embodiment, a composition useful for protection against infections caused by Variola Major is provided. The composition comprises at least one peptide comprising the sequence selected from the group consisting of SSVSPGQGKDSP, DVDPPTTYY, GSNISHKKVSYED, LYDLQRSAMVY, YDLQRSAMV, YRDKLVGKTVK, PSTADLLSNY, STADLLSNY, SANEVAKKL, LKWRCPFPDQ, LSYSREQT, LSYSREQTM, LGIAHLLEHLLISF, IRFHIKELENEYYF,

MGFGCYLSHWKCVI, CLVSFYDSGGNIPT, VEVNQLLESECGMFI, WNPILPTCV, KTVCKSTQ, EIITLAKKY, VSDNERCYV, RVYEKDYAL, and conservative modifications thereof.

In yet another embodiment, a composition useful for protection against infections caused by Clostridium botulinum is provided. The composition comprises at least one peptide comprising the sequence selected from the group consisting of LAHELIHAGHRL, FEELRTFGGHD,

YNQYTEEEKNNI, HQFNNIAKL, VASNWYNRQ, IERSSRTLG, LMHELIHVLHGL, AEELYTFGGQD, YNIYSEKEKSNI, HRFYESGIVFEEY, KDYFCISKWYLK, EVKRKPYNLKLG, LMHELNHTMHNL, YAEIYAFGGPT, YKKYSGSDKENI, LMHELTHSLHQL, FEELYTFGGLD, YKKYSGSDKENI, LMHELIHSLHGL, IEEFLTFGGTD, YNSYTLEEKNEL, STWYYTHMRDH, LAHELIHALHGL, LEEFLTFGGQD,

YNNYTSDEKNRL, HSNNLVASSWY, YNNIRRNTSSNG, LMHELIHVLHGL, AEELYTFGGHD, YNRYSEEDKMNI, SQWYLRRIS, ENINKLRLG, WGKEEEKRW, PIPETLIA YRR, DTDRDGIPDEWE,

GQIDPSVS, and conservative modifications thereof. A composition useful for protection against infections caused by

Shigella spp. is also provided. The composition comprises at least one peptide comprising the sequence selected from the group consisting of

DRLWHGFVVD, WHFATFNLAD, GYGDVKFLAA, SMVDKDWNNS, SDTDKHYQTE, NNLHTGESIK, HEKNVMNDAW, HTDVVPPGDA, EEASAHNGTV, NATIHKINEC, YNYHGKHEF, HVDTSPDCSG,

EEVGKGAKHF, DGGGVGELEF, TMTEEAGMDGA, GPTITGPHSP,

GGRFNGGHASH, and conservative modifications thereof.

A composition useful for protection against infections caused by both Bacillus anthracis and Yersinia pestis is provided. The composition comprises at least one peptide comprising the sequence selected from the group consisting of HFLEHM, HYLEHM, TTLGADNGI, GLKGGHSG, NAIPRE, GWKVKQG, GWKVKRG, IHECGHA VYEQNI, VHETGHARYEQNL IHESQSL RITTRY LHEIAHGYQ, GNSGGAL, EGIGLAIP, IGIGFAIP, PASMTKIM, PASLTKIM, SGNDAS, MRVISVV, MRLISVV, SMMPTL, DYVKRIIGLPGD, DYIKRVVGLPGD,

FVHGGPG, FIHGGPG, GFFLNN, GSPGGNRI, GSPGGSRI, AYSGRC, SLKEGR, SLKIEGR, LKFSDGSPLTA, KAGLDPE, AHNDEMALGA, AHNDDMAIGA, LKPDLII, LKPDVII, QIEQGAPADLF, and conservative modifications thereof. A composition useful for protection against infections caused by

Helicobacter pylori is provided. The composition comprises at least one peptide comprising the sequence selected from the group consisting of GIDVHSSGV, MFPDGTKLV, IHLSTQANV, KLENINKDL, KEANRDARA, VKGTDFFNV, HKATEEIYE, RTDDKNESY, MKHGIALV, KTKSNIEL, RNLAKSVTV, VDIVDGGTM, STGPHLHFG, GSFVKKGQI, YVKRNFAIG, LIDKISTIK, GNCVVKANA, QLNAATDAL, LSSGLRINV, KAYGKRMLY, SYQKDAKEL, VSAHEVSHGFTEQNS, KEKGIKLNYVEAVAL, GTAVRFEPGEEKSVE, GDTHIHFISPQQIPT, GFKIHEDWGTTPSAI, ITSSDSQAMGRVGEV,

AIHTDTLNEAGCVED, KLNESKSGNKNKMEA, DFLSSNKELVGKAVA, LAERIVQLGHHPLVT, KEGRHYRIDDGTPSD, FPLVISGINLGSNMG, VSIAHTRWATHGKP, IGVHLAHYLKRNFS, ITWAGSAHEVEMLHT IPSRSMVGTLYEGDM, LSGFAGSTADAFSLF, IGSGGNYALSAARAL, PRKLVEESLKIAGDL, AFLNGNVEAIVGGFG,

KMLDKVRSDLGSVQN, AMSQANTVQQNILRL, VVLYFYPKDNTPGCT, KCFYNVKAKGHAQKV,

IETNEVALKLNYHPA, and conservative modifications thereof.

Finally, a composition useful for protection against infections caused by Chlamydia pneumoniae is provided. The composition comprises at least one peptide comprising the sequence selected from the group consisting of

IFYGHIVDF, RRVHRIQQL, AHVLEHMVL, KIVASAETK, AVAESLITK, GDYVFDRIL, AVDRPNPAY, YVFDRILKVDAPKTF, ATANYTTAVDRPNPA, HGLENLYHV, TYELIKALL, SYGGVVPEL, EAHLYAAYMA, DIIEKAIDR, TVVTGLAAS, IFSPEKAKQE,

IVTQNRPLLTKEKLV, TSYLSDDIH, GSDNKGQ, QEDLDREKYAVHQE, AVFHPLLTKQSFLQE, RNKQPVEQKSRGAF, SVKVNDDCNVEICQS, FELYEVPIGSMRPTI, KYFGLIPGKKRYIKR, KGNNSSPRSPAPEISL, QPRDIIEKAIDRDMV, YNHYDVQPAQLSDG, and conservative modifications thereof.

The invention will be described in greater detail in the following Examples.

EXAMPLE 1 Genomic Analysis of B. anthracis Secreted Proteins as Potential

Virulence Factors

A whole-genome level comparative analysis of all protease genes in the genomes of B. cereus, B. subtilis, and two virulent anthrax strains, using the known sequence motifs characteristic of hundreds of families of proteolytic enzymes, was performed. Some of the considered motifs included H-E-X-X-H, [S,G,A]-H-X-D-X-V, G-X-X-D, and E-E motifs (some of the motifs are shown in bold italics in Table 2 that follows).

The analysis showed that the proteases that are the most potent of the ones secreted by B. anthracis in culture are metallo-protease (MP) enzymes, which act primarily as collagenases. In addition, several closely related thermolysin-like MPs of the M4 family are candidate enzymes that are capable of causing a hemorrhagic effect similar to thermolysin (EC 3.4.24.27) from Bacillus thermoproteolyticus. Moreover, these MPs are more abundant in both B. anthracis and B. cereus, compared to B. subtilis.

An analysis of the chromosome sequence of the B. anthracis Ames strain was performed based on shared sequence homology with pathogenic factors in other bacterial species. (Supran et al, 2002; Read et ah, 2003) This analysis revealed a variety of potential virulence-enhancing factors, including collagenases, phospholipases, haemolysins, proteases, and other enterotoxins. In fact, the B. cereus group of bacteria, which are pathogenic to humans or insects and includes B. anthracis, B. thuringiensis, and B. cereus, has more sequences that are predicted to be secreted proteins than does nonpathogenic B. subtilis (Read et al, 2003). These B. cereus group- specific genes represent the adaptations to a pathogenic lifestyle by the common ancestor, which was quite similar to B. cereus.

The most interesting of the secreted proteins is the group of proteases encoded on the B. anthracis chromosome that are shared in common with B. cereus, but are absent or relatively rare in the genomes of nonpathogenic bacteria. A large number of these proteases fall into clan MA (classified according to the MEROPS system, Barrett AJ, 2004). This clan includes thermolysin-like enzymes of the M4 family and others. The metallo- proteases (MPs) from several bacterial species belonging to this family are capable of causing massive internal hemorrhages and other life-threatening pathologies (Supuran et al, 2002; Sakata et al, 1996; Shin et al, 1996;

Miyoshi et al, 1998; Okamoto et al, 1997).

Eleven protease families are present in B. anthracis and B. cereus, but absent in B. subtilis. Six of these eleven subfamilies encode MPs. Three of the MP subfamilies, namely the M6, M9B, and M20C subfamilies, are encoded on the bacterial chromosomes. Members of the M6 peptidase family are usually described as "immune inhibitors" because in B. thuringiensis they can inhibit the insect antibacterial response (Lovgren et al., 1990). The M20C peptidase subfamily represents exopeptidases (Biagini et al., 2001) that are the unlikely cause of tissue destruction or internal bleeding. The collagenolytic proteases of the M9B family have potential pathogenic functions.

This genomic analysis indicated that the M4 family of thermolysin/elastase-like neutral proteases and the M9 family of collagenases are virulence-enhancing factors of B. anthracis Ames strain.

EXAMPLE 2

Determination of Peptide Sequences for Multiple Pathogenic Organisms

Using the MEROPs database of peptidases, initial studies were conducted using the M4 and M9 peptidases from Bacillus anthracis, in which chemical inhibitors and rabbit immune sera against these peptidases displayed a protective efficacy in combination with antibiotic therapy. These studies suggested a multi-pathogen vaccine candidate including the M4 and M9 peptidases. The peptidase sequences were selected based on similarity to other pathogenic organisms, as represented by their inclusion in the same peptidase family and as seen from the multiple sequence alignment that showed the sequence conservation among the peptidases. Additionally, each peptidase family was screened for noted homologs in animals (including human) based on the taxonomic distribution chart included in the MEROPs summary records. Those that did not contain any homolog in human were allowed to proceed to the next step. Thus, the resulting list of peptidase sequences encompasses vaccine candidates that can target multiple pathogenic organisms, but in which the likelihood of immune complications in humans would be minimal.

The list of peptidase sequences, which contains selected peptidase families consisting of multiple pathogenic organisms and which do not have homologs in human, is described below. Each peptidase is represented by a short amino-acid stretch that spans or is in near proximity to an active site motif. Each sequence is 6-13 amino acid residues in length, which is sufficient to allow for antibody recognition. In addition, each sequence includes the active site residue(s) as an additional mechanism to inhibit peptidase function if blocked by the immune system (Table 1).

Table 1

Examples of specific and common sequences of proteases and protease homologues for vaccine and therapeutic candidates

Each peptidase sequence contains a list of organisms that have similar sequences to the selected peptidase, either as curated by the MEROPs database or retrieved from the NCBI Blink program, which is a pre- computed form of BLAST (Altschul et al., 1997), using the default parameters. The resulting collection of sequences, from multiple organisms that represent sequence similarity to the selected peptidase, was put into a multiple alignment program called MuItAHn (Corept, 1988) to verify sequence conservation among sequences in each group, especially in the region of the active site residue(s). The sequences were then compared by a BLAST search against the human Reference Sequence (RefSeq) collection of proteins (Pruitt et al., 2003) to ensure that they do not express significant homology to any human protein.

EXAMPLE 3

Determination of 3-Dimentional Structure of Chosen Vaccine Candidates

The MEROPs database offers a wealth of information on Bacillus peptidases, including sequence data, catalytic activity, active-site residues, known structural conformations (when available) and relevant literature pertaining to function. It is known that a large number of peptidases exist that are potential virulence or virulence enhancing factors of bacteria and can be used as promising targets for vaccine and antibody development. The group of metal loproteinases that are encoded on the Bacillus anthracis chromosome and plasmids, and are not over-represented in non-pathogenic bacteria were highlighted because many of them play a significant role in the pathogenesis of many bacterial infections. For example, some metalloproteinases of the MA clan from several bacterial species, such as the M4 and M9 families, are known to contribute in life-threatening pathologies (Miyoshi et al., 1998; Okamoto et al., 1997; Sakata et al., 1996; Shin et al., 1996; Supuran et al., 2002). The MA clan's M6 metalloproteinase is a known immune inhibitor in some other Bacillus species and M34 metalloproteinase is the lethal factor of Bacillus anthracis.

A metalloproteinase selection process began by screening all 85 subfamilies of metalloproteinases and selecting eight peptidases by using the following selection principles and methods. First, the taxonomic distribution chart for each Bacillus anthracis metallopeptidase family was screened and metallopeptidases that did not contain homologues in animals (including human) were chosen. Thus, immune complications as a result of cross- reactivity to similar human proteins were avoided. Additionally, the selected peptidase sequences were BLASTed against the human Reference Sequence (RefSeq) collection of proteins (Pruitt et al., 2003) to confirm that they do not express significant homology to any human protein (Altschul et al., 1997). Default parameters were used and results were scanned based on a threshold of 25% identity if the selected peptidase matched a human protein. From this analysis, no selected peptidase sequence showed a significant similarity above the identity threshold to any human protein.

Combined with the above computational analysis performed for a potential target peptidase, the existing literature was reviewed and peptidases that are known to play a role in anthrax pathogenesis, such as M34 (anthrax lethal factor), or those that possess convincing evidence of their role in pathogenesis were chosen. In addition, the localization of the peptidase in relation to the bacterial cell were studied and peptidases that were either membrane-bound or extracellular were chosen, such that these peptidases can be easily accessible to the human immune system. Thus, from numerous Bacillus anthracis 's metal loproteinases in the MEROPs database, several peptidases were identified to be potential targets designated for further analysis.

A structural analysis of the selected peptidase sequences was then performed to determine the tertiary conformation of the peptidase. In particular, the location of the active site region in each selected peptidase was noted because it would serve as the best region to be targeted by the immune system (e.g., annihilation of peptidase functions if blocked by an antibody). For each peptidase, a target region that consisted of from six to thirteen amino-acids that spans the active site motif or is within proximity of the active site motif, which is sufficient to allow for antibody recognition, was considered. In addition, localization of the peptidase target regions was checked, for any residue within the region, to see whether it is placed on the surface of the peptidase. This was performed to clarify whether the target region would be accessible to the outside and capable of interacting with the human immune system and mediating specific immune response.

Structure data from the MEROPs records (if they were available) for any of the selected peptidases were collected. In cases where the MEROPs database could not provide a structural representation of a selected peptidase sequence, a conserved domain (CDD) search was performed using RPS- BLAST (default parameters, Marchler-Bauer et al., 2004), and structural representations were presented when available from a given Domain record (Marchler-Bauer et al, 2003). If no structure representations were available for a peptidase of interest in both the MEROPs database and the Domains database, then a Blink search (pre-computed with default parameters) was performed for structure records that showed amino acid sequence similarity to the selected peptidase sequence (Wheeler et al., 2004). If a similar structure record was found in Blink, then the structure record was visually displayed along with its amino acid region aligned to the selected peptidase sequence, thereby, inferring the structural configuration of the selected peptidase.

When structure records were not found from any of the above methods for a peptidase sequence, then the selected peptidase sequence was used in structure prediction analysis tools that allocate predicted chemical characteristics, such as solvent accessibility plots, and/or show similarity to existing structure records. Two such programs were used for this purpose: PredictProtein and PSIPRED. The PredictProtein program was used to detect chemical properties attributable to the selected peptidases, such as solvent accessibility (which infers localization of the sequence to the outside region of the protein) (Rost et al., 1996, and Rost et al., 1994). The PSIPRED protein structure prediction server provides structure predictions based on one of three methods; the one that was used in this case was GenTHREADER, a sequence profile based fold recognition method (McGuffin et al., 2000). Next, for each selected peptidase sequence from the group of potential targets, structure data was retrieved from curated databases and pre- computed analysis. In addition, structure data was retrieved from structure prediction tools, in order to deduce the 3D conformation of the peptidase and infer the placement of the amino acid residues - specifically the target region encompassing the active site residues - relative to the peptidase (Fig.3).

Structure records were available from MEROPs and the Domains database for M4, M32, and M34. M4 was represented by the structure of neutral proteinase from B. cereus. M32, which is a carboxypeptidase, was annotated in the Domains record with the structure of carboxypeptidase Apo- Yb from Pyrococcus furiosus. The structure of anthrax lethal factor was determined and represented in the MEROPs record for M34. Blink pre- computed results for M42, a glutamyl aminopeptidase, showed amino acid similarity to the structure record of aminopeptidase/glucanase from B. subtilis. PSIPRED and PredictProtein analyses were performed on the remaining peptidases M6, M9, Ml 5, and M60, for which structure records that were similar in amino acid sequence were also displayed. For all but two peptidases, additional confirmation on surface location of the target region was depicted using the Predict Protein program. Table 2 details the structure records that represent the confirmed or inferred structural representation for each selected peptide candidate for B. Anthracis derived from initial bioinformatics screening. The structure records were displayed using the program Rasmol (Sayle and Milner- White, 1995). The structural representation of the target region and the description of metal loprotease function (putative or experimental) are shown in Fig. 3.

The highlighted region, shaded light grey, represents the alignment of the structure's amino acid residues to the peptidase amino acid residues from the target region. The dark grey residues are annotated as surface residues, according to the Rasmol program. Table 2 highlights the selected metalloproteinases of Bacillus anthracis and selected specific peptide sequences of these metalloproteinases and a short description of the computational approach used to select them. Each chosen metalloproteinase shows no significant homology to any human protein. There is evidence of a presumed role in anthrax pathogenesis. Each target sequence region is determined or predicted to be localized to the outside of the protein and the bacterial cell, which is important for human immune recognition. The structural representations for each peptidase are listed, with reference to the source used to derive the structure records; and where suitable, identity scores are shown to reflect the significance of the amino acid similarity between a structure record and the peptidase sequence. Table 2.

Sequences of vaccine candidates for Bacillus anthracis derived from initial bioinformatics screening.

EXAMPLE 4

Generation of Antibodies against β. anthracis MPs.

Obvious complexity of the B. anthracis culture supernatant ("BACS") protein composition required the development of specific means of detection and inhibition of its components. Accordingly, several immune sera were raised in mice and rabbits using the antigens listed in Table 3. The sera were used in Western blots of BACS proteins. Table 3 Sera against B. anthracis proteases

When the proteins were directly separated in the SDS-PAGE for subsequent transfer to the nitrocellulose membrane, the resulting blots were of low intensity, indicating that proteolytic degradation had occurred during electrophoresis (Fig. IA, left lane). In order to avoid this complication, the BACS was fractionated according to the molecular masses of its components on the Superdex size exclusion column in the presence of EDTA as a chelating agent.

Analysis of the column fractions in SDS-PAGE showed a complex pattern of proteins bands (Fig. 1). Multiple proteins with a broad spectrum of molecular masses seem to be highly associated and migrate through the column as high molecular mass complexes. Several factors, such as the presence of multiple precursor and mature protein forms resulting from specific proteolytic maturation, along with nonspecific proteolytic products, can potentially contribute to the complexity of the composition of the fractions. Western blot experiments with column fractions revealed several discrete bands recognized by antibodies (Fig. 1). The M4 proteases are represented by several bands at about 50 KDa, as well as by the bands at about 40 and 20 KDa. These bands probably correspond to different maturation forms of proteases, including the enzymes lacking signal peptides, and mature enzyme forms. The M9 collagenases are detected as a band with a molecular mass of about 98 kDa, which is close to the estimated mass of the pro-enzymes, however the major gelatinase enzymatic activity corresponds to the 55 kDa proteins in the BACS.

EXAMPLE 5

Protection of Mice against β. anthracis Using Anti-Protease Sera

In order to evaluate a pathogenic potential attributed to the B. anthracis proteins other than known lethal and edema toxins, a nontoxigenic and nonencapsulated strain of B. anthracis (delta Ames), which is a parental Ames strain cured of both plasmids, pXOl and pXO2, was used. Mice were challenged intraperotineally (i.p.) with about 30 LD50 of B. anthracis Sterne spores. Treatment with a single daily dose of ciprofloxacin (50 mg/kg, i.p.) began at 24 h post challenge and continued for 10 days. The immune sera (each pulled from two rabbits) were administered once daily at a concentration of 25 mg/ml (i.p.). The sera displayed substantial differences in their protective effect (Fig. 2). The anti-M4 serum against the epitope(s) of the active center displayed the highest protection (60%), while the anti-collagenase serum (a-M9Coll) protected 30% mice. The anti-M4EP serum behaved similar to the naϊve serum. Both latter sera demonstrated no statistically reliable difference in survival, compared to untreated mice (10%, p>0.05). A combination treatment with both antibiotic and all studied immune sera, administered at the same dose (25 mg/kg) was synergistic and protected from 80 to 100% mice. A lower serum dose (5 mg/kg) showed a similar pattern of protection, however the effect of combination treatment was reduced to 70%.

The sequence of the M4EP peptide is ADYTRGQGIETY. The sequence of the M4AC peptide is DVIGHELTHAVTE. The sequence of the M9Coll peptide is HEFTHYLQGRYEVPGL.

EXAMPLE 6

Protection of Mice Against B. anthracis Using Peptide Vaccine

DBA2 female mice, 9-10 weeks old were immunized with combinations of peptides (see table 4 below) conjugated to KLH (105ug per mouse total) subcutaneously. One boost was given 2 weeks from initial immunization (100, 140, 160, or 165 ug per mouse) subcutaneously. The mice were challenged 3 weeks from the initial immunization with Sterne Strain of B. anthracis, 5x10⁷ spores per mouse, intraperotineally (i.p.). KLH was used as a non-vaccinated control because there is no difference in the mortality curve between control animals and those injected with KLH alone. Table 4. Peptide Combinations and Dosages:

Figure 4 shows that after only 3 weeks the peptides have induced an immune response and produce statistically significant protection (p=0.0003). The peptides used for immunization were against the proteases mentioned above and did not include some of the known virulence factors such as protective antigen or edema factor.

In addition, the combinations presented in the table 5 were also found effective against anthrax or plague in mice with the same immunization schedule described above.

Table 5. Peptides combinations.

EXAMPLE 7

Protection of Mice Against F. tularensis Using Peptide Vaccine

BALB/C female mice, 6 weeks old were immunized with individual peptides (10 ug per mouse total) or combination of peptides conjugated to KLH (60 ug per mouse total). The individual peptides were as follows: S14 (ETIVKDTDRD); M16 (DELNSIVENN); A24 (GYGDVKFLAA); AcpA (DAMSTNKFGV); C59 (KPQFTSYSVVD); and S49 (LIDKISTIK). The following mixtures of peptides were tested: T.I (DELNSIVENN, TSMVHEPNFD, EGHLLSPLLD, NRLMTPASTN, ETIVKDTDRD, YWFGKDALEL), T.2 (NRLMTPASTN, ETIVKDTDRD,

YWFGKDALEL, IHLSVQANAV, THFGEHPSLKI, and DAMSTNKFGV) and T.3 (DELNSIVENN, TSMVHEPNFD, EGHLLSPLLD, IHLSVQANAV, THFGEHPSLKI, DAMSTNKFGV).

Multi-sites subcutaneous injection was performed with a volume of less than lOOμl for each site, and at least 2 cm apart between each injection site. Injections between priming and first boost were spaced at 2 week intervals. The KLH-conjugated peptides were mixed with the adjuvant to form different combinations immediately before injection. The mice were challenged 4 weeks from the initial immunization with lOLDso of Francisella tularensis (LVS), intraperitoneal^ (i.p.). Figure 5 shows that after only 4 weeks the individual peptides as well as combinations of peptides have induced an immune response and produce a range of protection from 70% to 100%.

EXAMPLE 8 Treatment of Anthrax and Plague Using Anti-M32 and Anti-M60

Antibodies

Sequences were identified in both B. anthracis and Y. pestis that were very close in identity for M32 and M60. The sequences identified in anthrax for M32 and M60 are FGTIHECGHAVY and GTLHEIAHGYQA, respectively. The sequences identified in plague for M32 and M60 are

MGIVHETGHARY and WGCLHEIAHGYQ, respectively. Rabbit antibodies were produced to the plague sequence of M32 and the anthrax sequence of M60. The antibodies were tested against both pathologies individually, individually with ciprofloxacin, in combination, and in combination with ciprofloxacin. Antibodies were used in a concentration of

10 mg/kg for anthrax, 20 mg/kg for plague, and the concentration of ciprofloxacin was 50 mg/kg. Mice were infected with either anthrax or plague with treatment starting after 24 hours and lasting for 10 days. The concentration of B. anthracis Sterne strain was 5 x 10⁷ spores/mouse and Y. pestis KIM5-3001.1 was 6 x 10⁷ organisms/mouse. The treatment for anthrax using a combination of M32 and M60 plus ciprofloxacin resulted in 100% survival. A survival of 100% was achieved in treating plague using M32 antibody alone. Each of the groups providing 100% survival had a p- value < 0.01 compared to untreated mice.

EXAMPLE 9 Treatment of Anthrax Using Combinations of Anti-protease Antibodies

Sequences comprising the active center of metalloproteases M4, M6, M32, M34, and M60 were identified (see table 1) and antibodies against these sequences were raised in rabbits. The antibodies were tested against anthrax individually and in combination with ciprofloxacin. Antibodies were used in a concentration of 10 mg/kg and the concentration of ciprofloxacin was 50 mg/kg. Mice were infected with B. anthracis Sterne strain with a challenge dose of 5 x 107 spores/mouse. Treatment was initiated 24 hours after infection and continued for 10 days. Figure 7A demonstrates 60% survival using anti-M32 antibodies in combination with ciprofloxacin versus 10% survival of ciprofloxacin only treated mice. Figure 7B demonstreates

60% suruvival using anti-M60 antibodies in combination with ciprofloxacin versus 10% survival of ciprofloxacin only treated mice. Figure 7C demonstrates 100% survival using a combination of anti-M4, anti-M6, anti- M32, and anti-M34 antibodies in combination with ciprofloxacin versus 0% survival of ciprofloxacin only treated mice.

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Claims

What is claimed is:

I . A method of developing vaccines, antibodies and other inhibitors of pathogens to be used in subjects in need thereof, comprising:

(a) identifying at least one pathogen of interest; (b) obtaining a sequence of at least one protein produced by the pathogen;

(c) identifying at least one target region of the sequence comprising an allosteric binding site or an active site of the protein;

(d) selecting at least one portion of the target region that is accessible by a solvent to obtain at least one candidate sequence;

(e) synthesizing candidate peptides having the candidate sequences; and

(f) screening the candidate peptides for an immunogenic activity to identify at least one immunogenic peptide.

2. The method of claim 1, further comprising administering the immunogenic peptide or peptides to an animal to raise antibodies.

3. The method of claim 2, wherein the antibodies are monoclonal or polyclonal antibodies.

4. The method of claim 1, wherein the pathogen is an agent of biological warfare.

5. The method of claim 1, wherein the pathogen causes cardiac disease or cancer.

6. The method of claim 1, wherein step (a) comprises selecting a plurality of pathogens of interest and the target regions of step (c) contain regions conserved in the plurality of pathogens.

7. The method of claim 1, wherein the proteins produced by the pathogen are involved in anabolic or catabolic metabolism of the pathogen.

8. The method of claim 1, wherein the proteins produced by the pathogen are selected from a group consisting of secreted, membrane-bound, periplasmic proteases, and their homologues.

9. The method of claim 8, wherein proteases are metalloproteases.

10. The method of claim 8, wherein the proteases and their homologues are known or putative virulence or virulence-enhancing factors.

I 1. The method of claim 10, wherein the pathogen is B. anthracis and the virulence factors are selected from the group consisting of anthrax protective antigen (PA), lethal factor (LF), and edema factor (EF).

12. The method of claim 1, wherein the active site comprises HEXXH or EE motif.

13. The method of claim 1, wherein the candidate sequence is known or predicted to be located on an outer surface of a tertiary structure of the protein.

14. The method of claim 1, wherein step (d) further comprises comparing the candidate sequences with the animal protein sequences when the subject is an animal or with the human protein sequences when the subject is a human and excluding the candidate sequences having a predetermined level of homology with the animal or human proteins,respectively, from further method steps.

15. The method of claim 14, wherein the predetermined level of homology is at least 25%.

16. The method of claim 1, wherein the candidate sequences are at least 6 amino acids long.

17. The method of claim 1, wherein candidate sequences are less than 100 amino acids long.

18. The method of claim 1, wherein step (e) further comprises conjugating each of the candidate peptides to a carrier protein to obtain conjugated peptides and step (f) comprises screening the conjugated polypeptides for an immunogenic activity.

19. The method of claim 18, wherein the carrier protein is Keyhole Limpet Hemocyanin (KLH).

20. The method of claim 1 further comprising developing monopathogen, multipathogen, or monopathogen and multipathogen vaccines comprising a plurality of immunogenic peptides.

21. The method of claim 20, wherein the plurality of immunogenic peptides is used to raise specific antibodies.

22. The method of claim 1 further comprising comparing the sequence of the immunogenic peptides with the genomes of other pathogens to identify additional potential target regions.

23. A composition comprising (i) at least one immunogenic polypeptide of claim 1 or 18 or (ii) at least one antibody raised against at least one immunogenic polypeptide of claim 1 or 18.

24. A composition useful for protection against infections caused by B. anthracis comprising at least one peptide comprising the sequence selected from the group consisting of GTLHEI AHGYQ A, DVIGHELTHAVT,

GAVGVFAHEYGH, ELFRHEFTHYLQ, VIGHELTHAVTE, EYDTQYSGHGE, ELFRHEFTHYLQ, SAIPGTSEHQT, LILFEPANFNSM, DVIGHELTHAVTE, FGTIHECGHAVY, EGFIHEFGHAVD, EEVGLRGAKTSAN, ADYTRGQGIETY,

MGIVHETGHARY, VAGHLDEVGFMI, WGCLHEIAHGYQ, EEVGLRGAKTS, LVSIAGPISN, WGTLHEIAHGYQAG, WGTLHEIAHGYQA, HRMKDGSMFGS, VHGLNDWNVK, WLHQGGHGG, EIDNGKKYYLL, YNDKLPLYIS, DGAVGVFAHEYGH, EGFIHEFGHAVDD, VPDRDNDGIPDS, FLSPWISNIH,

NEPNGNLQYQGK, DQQTSQNIKNQ, DKLPLYISNPNY, NSRKKRSTSAGPT, VYYEIGK, HQSIGSTLYNK, DHRTVLELYAP, AND conservative modifications thereof.

25. The composition of claim 24, wherein the composition comprises a combination of four peptides comprising sequences GTLHEIAHGYQA,

DVIGHELTHAVTE, VAGHLDEVGFMI or conservative modifications thereof, and a sequence selected from the group consisting of MGIVHETGHARY and FGTIHECGHAVYor conservative modifications thereof.

26. The composition of claim 25, wherein the composition further comprises peptides comprising sequences ELFRHEFTHYLQ, EGFIHEFGHAVD, or conservative modifications thereof, and a sequence selected from the group consisting of EYDTQYSGHGE, GAVGVFAHEYGH, and conservative modifications thereof.

27. The composition of claim 26, wherein the composition further comprises peptides comprising sequences SAIPGTSEHQT, GNMDDYDWMNEN, WVRAHDNDEHYM, or conservative modifications thereof, and a sequence selected from the group consisting of WGCLHEIAHGYQ, EEVGLRGAKTSAN, and conservative modifications thereof.

28. The composition of claim 25, wherein the composition further comprises peptides comprising sequences LILFEP ANFNSM, WGCLHEIAHGYQ, GNMDDYDWMNEN, WVRAHDNDEHYM, or conservative modifications thereof.

29. A composition useful for protection against infections caused by Yersinia pestis comprising at least one peptide comprising the sequence selected from the group consisting of FNLAD VAICIGAAL, NAGYYVTPNAKV, GRQTFTHEI, GHALGLSHP, SRHHWGSDLDI,

LMGIVHETGHARYE, MGIVHETGHARY, EMDIQRSSSECGIFS, SCEGSQFKLFD, SMGGMAASGGYWIS, AKGYWVENGEI, TTIQVDGSEKK, SQDGNNHQFTT, DPQWKYSQETA, DNFMKDVLRLIEQ, EQYVSSHTHAMK, SPYGPEARAELSSR, GNMDDYDWMNEN, WVRAHDNDEHYM, FNLADVAICIGAAL,

SRHHWGSDLDIYDP, WGCLHEIAHGYQGGF, LILFEPANFNSMG, and conservative modifications thereof.

30. A composition useful for protection against infections caused by Francisella tularensis comprising at least one peptide comprising the sequence selected from the group consisting of GYGDFKLLAA,

KPQFTSYSVVD, DELNSIVENN, TSMVHEPNFD, EGHLLSPLLD, TAYHEAGHAI, STGGSKMYVK, NRLMTPASTN, ETIVKDTDRD, YWFGKDALEL, EKLQTTHFSI, IHLSVQANAV, GKFSLDVDFT,

THFGEHPSLKI, DAMSTNKFGV, VGSQNSSNSNR, and conservative modifications thereof.

31. A composition useful for protection against infections caused by Variola Major comprising at least one peptide comprising the sequence selected from the group consisting of SSVSPGQGKDSP, DVDPPTTYY, GSNISHKKVSYED, LYDLQRSAMVY, YDLQRSAMV, YRDKLVGKTVK, PSTADLLSNY, STADLLSNY, SANEVAKKL,

LKWRCPFPDQ, LSYSREQT, LSYSREQTM, LGIAHLLEHLLISF, IRFHIKELENEYYF, MGFGCYLSHWKCVI, CLVSFYDSGGNIPT, VEVNQLLESECGMFI, WNPILPTCV, KTVCKSTQ, EIITLAKKY, VSDNERCYV, RVYEKDYAL, and conservative modifications thereof.

32. A composition useful for protection against infections caused by Clostridium botulinum comprising at least one peptide comprising the sequence selected from the group consisting of LAHELIHAGHRL,

FEELRTFGGHD, YNQYTEEEKNNI, HQFNNIAKL, VASNWYNRQ, IERSSRTLG, LMHELIHVLHGL, AEELYTFGGQD, YNIYSEKEKSNI,

HRFYESGIVFEEY, KDYFCISKWYLK, EVKRKPYNLKLG, LMHELNHTMHNL, YAEIYAFGGPT, YKKYSGSDKENI, LMHELTHSLHQL, FEELYTFGGLD, YKKYSGSDKENI, LMHELIHSLHGL, IEEFLTFGGTD, YNSYTLEEKNEL, STWYYTHMRDH, LAHELIHALHGL, LEEFLTFGGQD,

YNNYTSDEKNRL, HSNNLVASSWY, YNNIRRNTSSNG, LMHELIHVLHGL, AEELYTFGGHD, YNRYSEEDKMNI, SQWYLRRIS,

ENINKLRLG, WGKEEEKRW, PIPETLIAYRR, DTDRDGIPDEWE, GQIDPSVS, and conservative modifications thereof.

33. A composition useful for protection against infections caused by

Shigella spp. comprising at least one peptide comprising the sequence selected from the group consisting of DRL WHGF V VD, WHFATFNLAD,

GYGDVKFLAA, SMVDKDWNNS, SDTDKHYQTE, NNLHTGESIK, HEKNVMNDAW, HTDVVPPGDA, EEASAHNGTV, NATIHKINEC, YNYHGKHEF, HVDTSPDCSG, EEVGKGAKHF, DGGGVGELEF,

TMTEEAGMDGA, GPTITGPHSP, GGRFNGGHASH, and conservative modifications thereof.

34. A composition useful for protection against infections caused by Bacillus anthracis and Yersinia pestis comprising at least one peptide comprising the sequence selected from the group consisting of HFLEHM,

HYLEHM, TTLGADNGI, GLKGGHSG, NAIPRE, GWKVKQG, GWKVKRG, IHECGHAVYEQNI, VHETGHARYEQNL IHESQSL RITTRY LHEIAHGYQ, GNSGGAL, EGIGLAIP, IGIGFAIP, PASMTKIM, PASLTKIM, SGNDAS, MRVISVV, MRLISVV, SMMPTL, DYVKRIIGLPGD, DYIKRVVGLPGD, FVHGGPG, FIHGGPG, GFFLNN,

GSPGGNRI, GSPGGSRI, AYSGRC, SLKEGR, SLKIEGR, LKFSDGSPLTA, KAGLDPE, AHNDEMALGA, AHNDDMAIGA, LKPDLII, LKPDVII, QIEQGAPADLF, and conservative modifications thereof.

35. A composition useful for protection against infections caused by Helicobacter pylori comprising at least one peptide comprising the sequence selected from the group consisting of GIDVHSSGV, MFPDGTKLV, IHLSTQANV, KLENINKDL, KEANRDARA, VKGTDFFNV, HKATEEIYE, RTDDKNESY, MKHGIALV, KTKSNIEL, RNLAKSVTV,

VDIVDGGTM, STGPHLHFG, GSFVKKGQI, YVKRNFAIG, LIDKISTIK, GNCVVKANA, QLNAATDAL, LSSGLRINV, KAYGKRMLY, SYQKDAKEL, V SAHEV SHGFTEQNS, KEKGIKLNYVEAVAL, GTAVRFEPGEEKSVE, GDTHIHFISPQQIPT, GFKIHEDWGTTPSAI, ITSSDSQAMGRVGEV, AIHTDTLNEAGCVED,

KLNESKSGNKNKMEA, DFLSSNKELVGKAVA, LAERivQLGHHPLVT, KEGRHYRIDDGTPSD, FPLVISGΓNLGSNMG, VSIAHTRWATHGKP, IGVHLAHYLKRNFS, ITWAGSAHEVEMLHT IPSRSMVGTLYEGDM, LSGFAGSTADAFSLF, IGSGGNYALSAARAL, PRKLVEESLKIAGDL₅ AFLNGNVEAIVGGFG,

KMLDKVRSDLGSVQN, AMSQANTVQQNILRL, VVLYFYPKDNTPGCT, KCFYNVKAKGHAQKV,

IETNEV ALKLNYHPA, and conservative modifications thereof.

36. A composition useful for protection against infections caused by Chlamydia pneumoniae comprising at least one peptide comprising the sequence selected from the group consisting of IFYGHIVDF,

RRVHRIQQL, AHVLEHMVL, KIVASAETK, AVAESLITK, GDYVFDRIL, AVDRPNPAY, YVFDRILKVDAPKTF, ATANYTTAVDRPNPA, HGLENLYHV, TYELIKALL, SYGGVVPEL, EAHLYAAYMA, DIIEKAIDR, TVVTGLAAS, IFSPEKAKQE,

IVTQNRPLLTKEKLV, TSYLSDDIH, GSDNKGQ, QEDLDREKYAVHQE, AVFHPLLTKQSFLQE, RNKQPVEQKSRGAF,

SVKVNDDCNVEICQS, FELYEVPIGSMRPTI, KYFGLIPGKKRYIKR,

KGNNSSPRSPAPEISL, QPRDIIEKAIDRDMV, YNHYDVQPAQLSDG, and conservative modifications thereof.

37. A vaccine comprising one of the compositions of claims 24-36.

38. A composition useful for treatment infections caused by pathogens of claims 24-36, wherein the composition comprises at least one antibody raised against at least one peptide of claims 24-36.

39. The composition of claim 23 or 38, wherein the composition further comprises a pharmaceutically acceptable carrier.

40. The composition of claim 23 or 39 further comprising an additional active ingredient.

41. The composition of claim 40 wherein the composition comprises at least one antibody and the additional active ingredient is an antibiotic.

42. The composition of claim 41, wherein the antibiotic is ciprofloxacin.

43. A method of vaccinating a patient against a disease comprising:

(1) developing a vaccine comprising immunogenic peptide or peptides-according to the method of claim 1 ;

(2) administering the vaccine to the patient.

44. A method of treating a patient suffering from a disease comprising: (1) obtaining immunogenic peptides of claim 1 ;

(2) raising antibodies against the immunogenic peptide or peptides; (3) administering the antibodies to a patient, wherein the harmful effects of the pathogen or pathogens are alleviated.

45. The method of claim 43 or 44, wherein the disease is the result of biological warfare.

46. The method of claim 43 or 44, wherein the disease is cardiac disease or cancer.